Respiratory syncytial virus (RSV) infection accounts for substantial worldwide morbidity and some mortality among infants and elderly and those with cardiac or pulmonary issues. The standard of care for RSV-infected patients is limited to supportive therapy, and there is no safe and efficacious RSV vaccine. There is one approved anti-RSV drug, i.e. inhaled ribavirin, but its effectiveness is questionable and it is difficult to administer. The long-term goal of this project is to develop a novel class of RSV anti-viral therapeutics that target the host protein nuclear export machinery (Exportin-1/XPO1) that is co-opted by RSV for its replication. A prototypical inhibitor of XPO1, leptomycin B (LMB) has been shown in vitro to block RSV replication. However, LMB failed Phase 1 trials due to in vivo toxicity with anorexia and malaise. Karyopharm Therapeutics has developed first-in-class selective inhibitors of nuclear export which block XPO1-directed nuclear export. One compound, KPT-335 (Verdinexor), is in a late-phase development for treatment of canine malignancy. We hypothesize that KPT-335 will safely and effectively block RSV replication by inhibiting nuclear export of RSV M protein. As the nuclear export signal (NES) of the RSV M protein is conserved among strains, we propose therapeutic and prophylactic studies to assess KPT-335 efficacy against different RSV group A and B strains, and determine the mechanism of action against RSV by evaluating M protein localization and XPO1 binding to the M protein. The overall goal of these studies proposed is to achieve a critical milestone and establish in vitro anti-RSV efficacy of KPT-335 as the basis to move this important drug forward for prospective in vivo studies.